Complete chloroplast genome of Daphne pseudomezereum var. koreana (Thymelaeaceae)

Abstract Daphne pseudomezereum A. Gray var. koreana (Nakai) Hamaya is a shrub distributed in high mountains in Japan and Korea and is used as a medicinal plant. The complete chloroplast genome of D. pseudomezereum var. koreana is 171,152 bp long with four subregions consisting of a large single-copy region (84,963 bp), a small single-copy region (41,725 bp), and a pair of inverted repeats (2739 bp). The genome includes 139 genes (93 protein-coding genes, eight rRNAs, and 38 tRNAs). Phylogenetic analyses show that D. pseudomezereum var. koreana is nested within the Daphne clade in the narrow sense and that it forms a distinct lineage.


Introduction
Daphne pseudomezereum A. Gray var. koreana (Nakai) Hamaya (Bull. Tokyo Univ. Forest. 55: 72, 1959) is a shrub distributed in high mountains in Japan and Korea (Oh and Hong 2015). As a member of Thymelaeaceae, which includes many ecologically important species for ornamentals, timber, papermaking, and medicine (Herber 2003), D. pseudomezereum can also be used as a medicinal plant for chronic skin diseases and rheumatism (Konishi et al. 1993). Plants of D. pseudomezereum var. koreana have rarely been collected, as they are uncommon. For this reason, little is known about the species. Detailed and accurate information on its phylogenetic position and better knowledge of the genome of the species will be useful for understanding its genetic diversity and for evaluating its phytochemical composition for medicinal purposes.

Materials and methods
The sample of D. pseudomezereum var. koreana (Figure 1) were collected in Sangye-ri, Okgye-myeon, Gangneung-si, Gangwon-do, Republic of Korea (37 32 0 48 00 N, 128 51'31 00 E). A voucher specimen was deposited in the Daejeon University Herbarium (TUT: https://www.dju.ac.kr/biosci/depart/ profileView.do?mi=2253, contact person: Sang-Hun Oh, soh42@dju.kr) under the voucher number Lee 7891. Total DNA was isolated from fresh leaves of the species using a DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). A small portion (3 ul) of the extracted DNA was run in 1% agarose gel to examine the quality. The concentration of DNA was measured with an Invitrogen Qubit fluorometer. An amount of 1 microgram of DNA was used to reconstruct the sequencing library. We used an Illumina TruSeq Nano DNA Library Preparation Kit (Illumina, San Diego, CA) following the manufacturer's recommendations. The sequencing library was analyzed using NovaSeq6000 at Macrogen Inc., Korea. The resulting 7.34-Gbp raw sequences were filtered using the Trimmomatic tool (v0.33) (Bolger et al. 2014). The chloroplast genome was de novo assembled with Velvet v1.2.10 (Zerbino and Birney 2008) and gaps were closed using GapCloser v1.12 (Zhao et al. 2011). The genome sequence was confirmed by aligning all raw reads against the assembled genome using BWA v0.7.17 and SAMtools v1.9 (Li et al. 2009;Li 2013) in the environment of Genome Information System (GeIS; https://geis.infoboss.co.kr/). Geneious Prime V R v2020.2.4 (Biomatters Ltd., Auckland, New Zealand) was used for annotation based on the Daphne genkwa chloroplast (MT754180) (Yoo et al. 2021) and read coverage depth map (Supplementary Figure 1). A circular map of the chloroplast genome ( Figure 2) and a schematic map of the cis-and trans-splicing genes ( Supplementary Figures 2 and 3) were generated by CPGView (Liu et al. 2023).
In total, 35 whole chloroplast genomes of Daphne and its closely related species (Herber 2003) were included in phylogenetic analyses using the maximum likelihood (ML) and Bayesian inference (BI) methods to infer the phylogenetic position of the chloroplast genome of D. pseudomezereum var. koreana. Pimelea and Gonystylus were used as outgroups. A heuristic search was conducted with nearest-neighbor  yc f2 (• 0. 2 3) tr n V r r n 1 6 t r n I tr n A r r n 2 3 r r n 4 . 5 nd hG   interchange branch swapping, the Tamura-Nei model, and uniform rates among sites to construct the ML phylogenetic tree with default values for other options using MEGA X (Kumar et al. 2018). A bootstrap analysis with 1000 pseudoreplicates was also conducted with the same search options. The BI tree was constructed using MrBayes v3.2.6 (Ronquist et al. 2012). The GTR model with gamma rates was used as a molecular model. A Markov-chain Monte Carlo algorithm was employed for 1,000,000 generations, sampling trees every 200 generations, with four chains running simultaneously.

Results
The chloroplast genome of D. pseudomezereum var. koreana (GenBank accession: ON244034) is 171,152 bp long (Figure 2). The overall GC ratio is 36.5%. The genome has four subregions consisting of 84,963 bp of a large single-copy region (LSC; 49.6%) and 2739 bp of a small single-copy (SSC; 1.6%) region separated by 41,725 bp of each of two inverted repeat regions (IRs; 48.8%). It contains 139 genes (93 protein-coding genes, eight rRNAs, and 38 tRNAs); 28 genes (16 protein-coding gene, four rRNAs, and eight tRNAs) are duplicated in IR regions (Figure 2). The phylogenetic tree showed that D. pseudomezereum var. koreana is nested within the Daphne clade, forming a distinct lineage within the clade (Figure 3). It was sister to a large subclade that includes most species of Daphne, such as D. giraldii and D. tangutica (Figure 3).

Discussion and conclusion
The chloroplast genome of D. pseudomezereum var. koreana exhibits long IR regions and a short SSC region compared to a typical chloroplast genome of angiosperms (Palmer et al. 1988;Cosner et al. 2004). The expansion of IR associated with the shortening of SSC is also found in other species of Thymelaeaceae, such as Daphne kiusiana (Cho et al. 2018) and   It appears that the events of IR expansion and contraction should have occurred multiple times within Thymelaeaceae, and more chloroplast genomes for various taxa are needed to understand the pattern of chloroplast evolution in the family.
The phylogenetic tree indicates that the position of newly determined chloroplast genome of D. pseudomezereum var. koreana is consistent with morphology (Oh and Hong 2015; Lee J-J and Oh 2017)), placed within the core Daphne clade. The phylogenetic analysis suggests that the Daphne should be narrowly redefined to indicate the core Daphne clade (Figure 3), as the traditional circumscription of the genus Daphne (Herber 2003;Wang et al. 2007;Oh and Hong 2015) is polyphyletic. It also suggests that Wikstroemia should expand to include D. genkwa, D. aurantiaca, and Stellera chamaejasme and that D. championi is sister to Edgeworthia chrysantha (Figure 3). Our results, consistent with previous analyses (Yoo et al. 2021;Lee SY et al. 2022), indicate that further detailed systematic studies of Daphne and its closely related groups with more taxon sampling are needed. This report contributes to the understanding of the chloroplast genetic information of D. pseudomezereum var. koreana to provide additional data for future research to reconstruct the evolutionary relationships and for the establishment of a sound classification system of Daphne and its closely related groups and ultimately to develop molecular markers for the medicinal plant.

Ethical approval
The authors declare that there were no ethical or legal violations when obtaining the study materials and performing the research. The species used in this study is not listed on the IUCN Red List, and the sample was legally collected in accordance with guidelines stipulated in national and international regulations. The materials were collected in a location that was not designated as a protected area in Korea. No ethical approval or permission from the Institutional Review Board (IRB) of Daejeon University was required for this study.